Method for detecting low concentrations of a target bacterium that uses phages to infect target bacterial cells

ABSTRACT

The invention is directed to a method for detecting low concentrations of bacteria in liquid solution that may or may not be complex liquid solutions. In one embodiment, immunomagnetic separation (IMS) is used to separate target bacterium that may be in a liquid mixture from other constituents in the mixture. A low concentration of a bacteriophage for the target bacteria is subsequently used to infect target bacterial cells that have been captured using the IMS technique. If at least a certain concentration of target bacterium are present, the bacteriophage will multiply to a point that is detectable. Matrix assisted laser desorption ionization/time-of-flight-mass spectrometry (MALDI/TOF-MS) is then used to produce a mass spectrum that is analyzed to determine if one or more proteins associated with the bacteriophage are present, thereby indirectly indicating that target bacterium were present in the liquid mixture.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/319,184, entitled “METHOD OF DETECTING LOW CONCENTRATIONS OF A TARGETBACTERIA THAT USES PHAGES TO INFECT TARGET BACTERIAL CELLS” and filed byAngelo J. Madonna and Kent J. Voorhees on Apr. 12, 2002, whichapplication is incorporated by reference into this application in itsentirety.

FIELD OF THE INVENTION

The present invention relates to a method for detecting lowconcentrations of a target bacterium in a liquid mixture that usesbacteriophages to infect target bacterial cells.

BACKGROUND OF THE INVENTION

Standard microbiological methods have relied on substrate-based assaysto test for the presence of specific organisms (Bordner, et al. 1978).These techniques offer very high levels of selectivity but are hinderedby the requirement to first grow or cultivate pure cultures of thetargeted organism, which can take 24 hours or longer. This timeconstraint severely limits the effectiveness to provide a rapid responseto the presence of virulent strains of microorganisms.

Molecular biology techniques are quickly gaining acceptance as valuablealternatives to standard microbiological tests. In particular,serological methods have been widely employed to evaluate a host ofmatrices for targeted microorganisms (Kingsbury & Falkow 1985; Wyatt etal. 1992). These tests focus on using antibodies to first trap and thenseparate targeted organisms from other constituents in complicatedbiological mixtures. Once isolated, the captured organism can beconcentrated and detected by a variety of different techniques that donot require cultivating the biological analyte.

One very popular approach, termed immunomagnetic separation (IMS),involves immobilizing antibodies to spherical, micro-sized paramagneticbeads and using the antibody-coated beads to trap targetedmicroorganisms from liquid media. The beads are easily manipulated underthe influence of a magnetic field facilitating the retrieval andconcentration of targeted organisms. Moreover, the small size and shapeof the beads allow them to become evenly dispersed in the sample,accelerating the rate of interaction between bead and target. Thesefavorable characteristics lead to reductions in assay time and helpstreamline the analytical procedure making it more applicable for highersample throughput and automation.

Downstream detection methods previously used with IMS include ELISA(Cudjoe, et al. 1995), dot blot assay (Skjerve et al. 1990),electrochemiluminescence (Yu and Bruno 1996), and flow cytometry (Pyle,et al. 1999). Although these tests provide satisfactory results, theyare laborious to perform and give binary responses (yes/no) that arehighly susceptible to false-positive results due to cross-reactivitywith non-target analytes. Recently reported is a rapid method foridentifying specific bacteria from complex biological mixtures using IMScoupled to matrix-assisted laser desorption/ionization (MALDI)time-of-flight (TOF) mass spectrometry (MS)(Madonna et al. 2001). Thisapproach allowed a variety of matrices to be evaluated for the presenceof a Salmonella species within a total analysis time of 1 hour.Moreover, the developed procedure required little sample processing, wasrelatively easy to perform, and the molecular weight informationobtained made it possible to discriminate between signals from thetarget bacteria and signals from cross-reacted constituents.

MALDI-TOF-MS is a proven technique for identifying whole cellularmicroorganisms (Holland et al (1996); van Barr 2000; Madonna et al.2000). In principle, MALDI is a ‘fingerprinting’ technique where massspectra featuring varying distributions of protein signals aregenerated. The signature profiles that are produced, due to inherentdifferences in microbial proteomes, make it possible to discriminatebetween organisms down to the strain level (Arnold and Reilly 1998). TheMALDI-TOF technique coupled with IMS includes, in one embodiment, mixingimmunomagnetic beads specific to the target bacteria with the liquidmixture that may contain the target bacteria for a short incubationperiod (e.g., 20 min). Any target bacteria captured by the beads arewashed twice, re-suspended in deionized H₂O, and directly applied onto aMALDI sample probe. The target bacteria-bead complex is then overlaidwith a micro-volume of matrix solution and dried at room temperature.Irradiation of the resulting crystalline mass with a high intensitylaser promotes the liberation and ionization of intact cellular proteinsthat are subsequently detected by a TOF mass spectrometer. The resultingmass spectrum is then interrogated for definitive mass peaks thatsignify the presence of the target bacteria.

SUMMARY OF THE INVENTION

The invention is directed to a method for determining if a targetbacterium is present in a liquid solution when the target bacterium isor may be present in a low concentration that is at or near thedetection limit for a particular detection technology. As used herein,the term “target bacterium” refers to a specie of species of bacteria.In turn, the invention is applicable to situations in which it isdesirable to determine whether a target bacterium (e.g., E. coli) ispresent in a liquid solution when the number of target bacterium perunit volume of solution (i.e., the concentration of the targetbacterium) is or may be below the detection limit for a particulardetection technology. In some instances, a plurality of target bacteriummay be referred to as the target bacteria.

In one embodiment, the process comprises using a biologicalamplification procedure in which bacteriophages for the target bacteriumare applied to the liquid solution. (Bacteriophages are viruses thatinfect bacteria and in the process produce many progeny. Structurally,the bacteriophage consists of a protein shell (capsid) that encapsulatesthe viral nucleic acid. The capsid is constructed from repeating copiesof the same protein. Bacteriophages are able to infect specificbacterial cells and because of the multiplication of the geneticmaterial, the cells eventually burst releasing millions of copies of theoriginal phage.) The bacteriophages and any of the target bacteriumpresent in the liquid solution are allowed to incubate. During theincubation period, the bacteriophages will multiply by infecting targetbacterium present in the solution. More specifically, the bacteriophagereplicates numerous copies of itself in an infected target bacterium.Eventually, the infected target bacterium lyses and the replicated orprogeny bacteriophages are released into the liquid solution. Thesolution is then analyzed to determine if a biomarker for thebacteriophage is present, thereby indirectly indicating that the targetbacterium is present in the liquid solution. Possible analysistechniques comprise mass spectrometry techniques, such as MALDI-MS andelectro-spray ionization-MS techniques.

To assure that the detection of a biomarker for the bacteriophageindicates that the target bacterium is present in the liquid solution, aconcentration of the bacteriophage is applied to the liquid solutionthat is below the detection limit for the biomarker for thebacteriophage for whatever analysis technique is employed. This assuresthat if the biomarker for the bacteriophage is detected by the analysistechnique, the detectable concentration of the biomarker is attributableto the replication of the bacteriophage by the target bacterium presentin the liquid solution. In certain situations, the use of such aconcentration of bacteriophage has a multiplicity of infection (“MOI”)(i.e., ratio of infecting bacteriophages to target bacterium) that istoo low to rapidly produce a sufficient concentration of bacteriophagesor biomarkers for the bacteriophage for detection.

Another embodiment of the process addresses this problem by adding avery high concentration of the bacteriophage to the liquid solution,thereby assuring a high MOI. In this case, the concentration of thebacteriophage added to the solution may exceed the detection limit ofwhatever analysis technique is employed to detect the bacteriophage orbiomarker of the bacteriophage. Consequently, the process applies parentbacteriophage to the solution that can distinguished from any progenybacteriophage resulting from the infection of target bacterium in themixture. If the distinguishable progency bacteriophage or adistinguishable biomarker of the progeny bacteriophage are present, thisindicates that the target bacterium is present in the solution

In one embodiment, the parent bacteriophage (i.e., the bacteriophageinitially applied to the solution) are “tagged” so that whateveranalysis technique is employed is inherently capable of distinguishingthe parent bacteriophage or parent bacteriophage biomarkers from theprogeny bacteriophage or biomarkers for the progency bacteriophage. Forexample, if a mass spectral analysis technique is employed, the parentbacteriophage are “tagged” with a substance that alters or shifts themass spectrum of the parent bacteriophage relative to the progenybacteriophage, which will not inherit the “tag” from the parentbacteriophage. For example, a biotinylated bacteriophage can be employedas a parent bacteriophage and will have a different mass spectrum thanthe progeny bacteriophage produced by the biotinylated bacteriophageinfecting target bacterium present in the solution. Other “tags” can beemployed for other types of analytical techniques.

In another embodiment, the parent bacteriophage possesses acharacteristic that allows the parent bacteriophage to be separated fromthe progeny bacteriophage in the liquid solution prior to analysis,thereby assuring that most, if not all of the bacteriophages present inthe liquid solution after separation are progeny bacteriophage resultingfrom the replication of the parent bacteriophage by target bacteriapresent in the liquid solution. In one embodiment, the parentbacteriophages initially applied to the liquid solution are biotinylatedbacteriophages. Biotinylated bacteriophages are highly attracted tostrepavidin. This attraction is exploited to separate the biotinylatedbacteriophage from progeny bacteriophage resulting from replication ofthe biotinylated bacteriophage by target bacterium present in themixture.

In one embodiment, the biotinylated bacteriophage are attached to astrepavidin coated probe. Consequently, separation of the biotinylatedbacteriophage from the liquid solution after the incubation period isaccomplished by removing the probe from the liquid solution. In anotherembodiment, strepavidin-coated magnetic beads are applied to the liquidsolution. The beads are used to pick up the biotinylated bacteriophage.The beads are then separated from the liquid solution using a magnet. Inyet another, embodiment a strepavidin coated probe (e.g., a slide) isapplied to the liquid solution after the incubation period. Thebiotinylated bacteriophage adhere to the probe and then the probe isseparated from the liquid solution.

Yet a further embodiment of the invention recognizes that the liquidsolution in which the target bacterium may be present is or may be acomplex mixture that includes biological material that makes thedetection of the bacteriophage or biomarker for the bacteriophage moredifficult or reduces the reliability of the information provided by thedetection technology employed. For instance, when a mass spectrometrydetection methodology is employed, the complex mixture may produce asignal in which the biomarker associated with the bacteriophage isobscured or, stated differently, has a low signal-to-noise ratio. Toaddress this possibility, the liquid solution is subject to apurification step in which target bacterium that are present in theliquid solution are separated from the remainder of the solution. In oneembodiment, immuno-magnetic separation (“IMS”) is utilized to separatetarget bacterium present in the liquid solution from the remainder ofthe solution. In one particular embodiment, magnetic beads are coatedwith an antibody for the target bacterium. The antibodies pick up thetarget bacterium present in the liquid mixture and then a magnet is usedto separate the beads from the remainder of the liquid solution. Thebeads and any adhering target bacterium are then subjected to thebiological amplification process and analysis. It should be appreciatedthis purification step also addresses the possibility that feralversions of the bacteriophage may be present in the liquid solution andthat such versions could produce a false positive if the liquid solutionwas not subjected to a purification step.

If feral versions of the bacteriophage are not of concern, thepurification step can be implemented after the biological amplificationprocess. In this embodiment, the purification step involves separatingthe bacteriophages and the liquid solution, rather than separating thetarget bacterium and the liquid solution. In one embodiment, an IMS isused in which magnetic beads are coated with an antibody for thebacteriophage. The beads pick up the bacteriophages present in thesolution and then a magnet is used to separate the beads from theremainder of the solution.

In another embodiment of the invention, the analysis step comprisesusing MS/MS analysis to determine if a biomarker for the targetbacterium is present. The use of MS/MS analysis produces a highlyreliable indication of the presence of a biomarker for a targetbacterium. As a consequence, at least in some cases, the use of MS/MSanalysis renders the need for a purification step unnecessary.

In yet another embodiment, the invention is directed to a process fordetecting low concentrations of a target bacterium in complex mixtures.In one embodiment, the process comprises using an IMS procedure toisolate at least some of a target bacterium that may be present in aliquid mixture. The process further includes employing a biologicalamplification procedure in which a low titer or concentration ofbacteriophages for the target bacterium are applied to at least some ofthe target bacterium that has been isolated by the IMS procedure. Themixture of bacteriophages and any of the target bacterium that has beenisolated is allowed to incubate. If at least a certain concentration ofthe target bacterium is present, the bacteriophages will multiply duringthe incubation period such that a high titer or concentration ofbacteriophages will be present in the mixture and detectable byMALDI-TOF-MS analysis. If no or only a small number of the targetbacterium is present, there will be a low concentration ofbacteriophages present in the mixture that will not be reasonablydetectable by MALDI-TOF-MS analysis. Following incubation, aMALDI-TOF-MS analysis is performed on the incubated mixture ofbacteriophages and target bacterium. The resulting mass spectrum isanalyzed to determine if a protein that is associated with thebacteriophages is present. If the protein for the bacteriophage isdetected, then it can be concluded that at least a low concentration ofthe target bacterium is present in the mixture.

It should also be appreciated that the method of the invention iscapable of detecting low concentration of a target bacterium regardlessof the manner in which the bacterium was grown or propogated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts, for one embodiment of the invention, an immunomagneticbead that is used to isolate the target microorganism (antibodies notdrawn to scale);

FIG. 2 is a schematic representation of the immunomagnetic purficationstep used to isolate a target antigen (Escherichia coli) in oneembodiment of the invention;

FIG. 3 is a schematic representation of the bacteriophage amplificationstep for one embodiment of the invention;

FIG. 4 shows a typical MALDI-TOF mass spectrum obtained from a hightiter sample of MS2 in PBS; and

FIGS. 5A–5C illustrate how an embodiment of the invention was abledetect a biomarker of the bacteriophage indicative of presence of E.coli for decreasing concentrations of E. coli.

DETAILED DESCRIPTION

Generally, the invention relates to the use of a bacteriophage toindirectly detect the presence of a target bacterium in a liquidsolution where the concentration of the target bacterium is or is likelyto be near or below the detection limit for the particular detectiontechnology employed.

Bacteriophages are viruses that infect bacteria and in the process ofinfecting the bacteria produce many progeny. Structurally, thebacteriophage consists of a protein shell (capsid) that encapsulates theviral nucleic acid. The capsid is constructed from repeating copies ofthe same protein(s). Bacteriophages are able to infect specificbacterial cells and because of the multiplication of the number ofprogeny, the cells eventually burst releasing millions of copies of theoriginal phage. This infection process has been utilized to serve as abiomarker amplification step for detecting low concentrations of targetbacterial cells. For example, the capsid of the MS2 bacteriophagecontains 180 copies of a 13 kDa protein. This particular virusspecifically infects strains of Escherichia coli and is able to producebetween 10,000 to 20,000 copies of itself within 40 min after attachmentto the target bacterial cell. Essentially, one E. coli could be infectedwith MS2 resulting in the replication of the capsid protein(s) by afactor of 1.8×10⁶.

The results from matrix assisted laser desorption ionization/massspectrometry (MALDI/TOF) can be used to show the utility of theamplification step. MALDI-TOF-MS is a proven technique for identifyingwhole cellular microorganisms (Holland et al 1996; van Barr 2000;Madonna et al. 2000). In principle, MALDI is a ‘fingerprinting’technique where mass spectra featuring varying distributions of proteinsignals are generated. The signature profiles are produced due toinherent differences in microbial proteomes that make it possible todiscriminate between organisms down to the strain level (Arnold andReilly 1998).

In an experiment where the protein MALDI signal from target bacterialcells was too weak for detection, the addition of low levels (too low todetect by MALDI) of the appropriate phage to the target bacterial cellsafter about thirty minutes produced a detectable protein MALDI signalattributable to the phage capsid protein. Bacteriophages specific forother bacterial species typically have capsid proteins of differentmolecular weight and therefore give a different MALDI signal. Therefore,the procedure is applicable to a multitude of different bacterialspecies. Other detection technologies, such as ion mobilityspectrometry, optical spectroscopy, immuno techniques, chromatographictechniques and aptamer processes, are also feasible.

Generally, the process for detecting low concentrations of a targetbacterium in a complex liquid mixture that contains or is likely tocontain biological material other than the target bacterium comprisesprocessing the mixture or a portion thereof to produce a liquid mixture,solution or sample for analysis that, if at least a certainconcentration of the target bacterium is present in the mixture, adiscernable signal or indication thereof is produced. It should beunderstood that the terms “liquid solution” and “liquid mixture” referto the original solution or mixture that is the subject of the test andany liquid solutions or mixtures that, as a result of the application ofthe method, contain a portion of the original solution or mixture.

In one embodiment, the process comprises making a determination if a lowconcentration of a target bacterium is present in the liquid solution.This determination can be made by assuming that any of the targetbacterium that are present in the liquid solution are present in a lowconcentration. Alternatively, an assay can be performed to determine ifthe target bacterium is present in a concentration that reliably exceedsthe detection limit of whatever detection technology is being utilized.For instance, a mass spectrometry technique can be utilized. If the massspectrometry technique provides a reliable signal indicative of thepresence of the target bacterium in the liquid solution, no furthersteps needs to be taken. If, however, the mass spectrometry techniquedoes not provide a reliable signal indicative of the presence of thetarget bacterium in the liquid solution, then it can be concluded thatthe target bacterium may be present in the liquid solution but in aconcentration that below or near the detection limit of the massspectrometry technique. In this case, further steps are taken todetermine whether the target bacterium is present in the liquid solutionin a concentration that is under the detection limit of the massspectrometry technique.

The process involves a purification step that involves capturing thetarget bacterium that may be present in the mixture and separating anyof the captured bacterium from other biological material that may bepresent in the mixture. By separating any of the captured targetbacterium from other biological material that may be present in themixture, the portion of the subsequently produced mass spectrum signalassociated with other biological material present in the mixture isreduced. In one embodiment, an immumomagnetic separation (IMS) techniqueis used to capture and separate the target bacterium.

The process further comprises subjecting at least some of any of thecaptured and separated target bacterium to an amplification step inwhich the target bacterium are infected with a bacteriophage that isspecific to the target bacterium. If there is at least a certainconcentration of the target bacterium present, the bacteriophage willmultiply to a point that a biomarker associated with the bacteriophagewill be detectable using an analysis techique, such as MALDI/TOF-MS. Inthe case of MALDI/TOF-MS, a portion of the subsequently produced massspectral signal indicative of the presence of the bacteriophage will beincreased. If there is less than a particular concentration of thetarget bacterium present, the bacteriophage will not multiplysufficiently to be detectable in the mass spectrum produced usingMALDI/TOF-MS. In essence, provided there is a least a certainconcentration of the target bacterium present in the mixture, thepurification and amplification steps serve to increase thesignal-to-noise ratio for the portion of the subsequently produced massspectrum that is associated with the bacteriophage.

After amplification, at least a portion of the amplified mixture issubjected to analysis to determine if a biomarker for the targetbacterium is present. For example, MALDI/TOF-MS analysis can be used toproduce a mass spectrum. The mass spectrum is analyzed to determine ifone or more biomarkers for the bacteriophage are present. If suchbiomarkers are present, this is an indirect indication that at least acertain concentration of the target bacterium was present in theoriginally sampled mixture.

It should be appreciated that the need for the purification step may notbe necessary in situations in which the portion of the signal associatedwith other biological materials can be filtered, eliminated or otherwiseignored and/or in situations in which the amplification step has a gainsuch that the portion of the mass spectrum associated with thebacteriophage is likely to exceed any background signal associated withother biological materials and/or situations in which a false positiveis considered to be remote. Further, it should also be appreciated thatone of the other biological materials that can make it difficult todetermine if the biomarker for the bacteriophage is present is a wildversion of the bacteriophage that is present in the liquid solution thatis being tested. The purification step addresses the presence of a wildbacteriophage.

If the presence or possible presence of a wild bacteriophage in thesolution or mixture being tested is not a concern, it is also possibleto perform a purification step after the amplification step. However, inthis case, the bacteriophage is separated from the remainder of theliquid solution. The previously noted IMS technique can be employed.However, the magnetic bead is coated with an antibody for thebacteriophage, rather than an antibody for the target bacterium.

It is also possible in certain cases to eliminate the need for apurification step by utilizing a highly specific analysis technique,such as MS/MS, in which the biomarker for the bacteriophage is so highlyspecific to the bacteriophage that there is little likelihood of a falsepositive. Currently, the use of MS/MS is limited to capsid proteins thatare less than 7000 Da.W

With reference to FIG. 1, the immunomagnetic separation (IMS) techniquefor capturing the target bacterium in a mixture and separating anycaptured target bacterium from other biological material in the mixtureis described. Microsize beads are constructed from an iron oxide corecoated with a polymeric surface. Secondary antibodies raised against theFc region of the primary antibodies are covalently attached via a linkerto the polymer surface. The primary antibody (raised against a targetedmicroorganism) is attached to the beads by strong noncovalentinteractions with the secondary antibody, which holds the primaryantibody in the proper orientation for reaction with the targetedantigen.

With reference to FIG. 2, the immunomagnetic beads are added to thebacterial or biological mixture that is the subject of the analysis andincubated for 20 minutes at room temperature. The beads are thenisolated to the side of the reaction tube using a magnet. This processallows the extraneous (non-targeted) material to be removed byaspiration. At this stage, the beads can be washed several times priorto re-suspending them in PBS.

With reference to FIG. 3, the bead-bacterial target complex is admixedwith a low titer suspension of bacteriophage specific to the targetedbacterium. The titer is held low so that the mass spectrometry signalfrom the virus is non-detectable. After a 40-minute incubation, thebacteriophage have completed a propagation cycle of attachment,insertion, self-assembly, and cell lysis resulting in the production ofmany progeny that are released into the reaction milieu. The milieu isthen analyzed to determine if a biomarker for the bacteriophage ispresent that indirectly indicates that the target bacterium was present.For instance, the milieu can be analyzed by MALDI-TOF-MS using asandwich sample preparation technique with a ferulic acid matrix. OtherMALDI matrices known in the art are also feasible. The resulting massspectrum shows the presence of the bacteriophage capsid protein, whichwould not have been present if the target bacterium was not alsopresent.

An IMS technique for capturing target bacterium in a mixture andseparating any captured target bacterium from other biological materialin the mixture and subsequent MALDI-TOF/MS analysis are described inU.S. patent application Ser. No. 10/063,346, entitled “Method forDetermining if a Type of Bacteria is Present in a Mixture,” filed onApr. 12, 2002, which is incorporated herein, in its entirety, byreference.

EXAMPLE

As described hereinafter, an embodiment of the method has been used toreduce the detection limit for E. coli to less than 5.0×10⁴ cells mL⁻¹.The method used immunomagnetic beads coated with antibodies against E.coli, hereinafter referred to as the target-bead complex, to isolate thebacterium from solution. The target-bead complex was then re-suspendedin a solution containing MS2, a bacteriophage that is specific for E.coli. The MS2 bacteriophage concentration was adjusted so that the ionsignal from the capsid protein of the MS2 bacteriophage was below thedetection limit of the mass spectrometer. After a 40-minute incubationperiod, an aliquot of the solution was removed and analyzed by theon-probe MALDI-TOF-MS procedure for the 13 kDa capsid protein. The[M+H]⁺ (m/z 13,726) and [M+2H]⁺² (m/z 6865) ion signals for the MS2capsid protein were detected (FIG. 4).

With reference to FIG. 5A, application of the process to a mixture thatcontains a concentration of 5.0×10⁶ E. coli cells mL⁻¹ yields a massspectrum with protein signals for both E. coli and the MS2bacteriophage. The process was repeated for decreasing concentrations ofE. coli. Specifically, with reference to FIG. 5B, the process wasrepeated for a concentration of ˜5.0×10⁵ E. coli cells mL⁻¹. In thiscase, the mass spectrum fails to definitively show any protein signalsfor the E. coli cells, but does show the protein signals for the MS2bacteriophage capsid protein. With reference to FIG. 5C, the process wasrepeated for an E. coli concentration of ˜5.0×10⁴ cells mL⁻¹. In thiscase, the mass spectrum fails to definitively show any protein signalsfor the E. coli cells but still show protein signals for the MS2bacteriophage capsid protein. These results indicate that E. coli wastrapped by the immunomagnetic beads and then infected by the MS2 virus,which was able to multiply and increase the concentration of the capsidprotein to a detectable level. Presently, target bacteriumconcentrations of as low as ˜1.0×10³ cells mL⁻¹ have been indirectlydetected using this process.

The following describes various aspects of the embodiment of the methodimplemented with respect to the example of the detection of E. coli.

E coli Preparation

The E. coli bacteria were grown in trypticase soy broth (TSB) (Difco,Detroit, Mich.) with incubation at 37° C. using standard microbialmethods.

Bacteriophage Propagation

Bacteriophage propagation was performed in accordance to the Adamsagar-overlay method as described in M. H. Adams' Bacteriophages(Interscience Publishers, Inc., New York, 1959). Briefly, asoft-agar/host covering was prepared by overlaying agar plates(trypticase soy agar, Difco) with a 2.5 mL of melted 0.5% agar (samemedium), which contained two drops of a 20 hr host in TSB. The soft-agarcovering was allowed to harden before the addition of a 0.5 mL overlayof a concentrated suspension of MS2, prepared by re-hydratingfreeze-dried MS2 in TSB. After 24 hours, the soft agar was scraped offthe surface of the agar plates and centrifuged (1000 G) for 25 min tosediment the cellular debris and agar. The supernatant was conserved,passed through 0.22 μm Millipore filters, and stored by refrigeration at4–8° C.

Immunomagnetic Bead Preparation R

Rabbit anti-E. coli IgG antibodies (Cortex Biochem, San Leandro, Calif.)were attached to the immunomagnetic beads (MagaBeads, Goat anti-RabbitIgG F(c), Cortex Biochem) using the manufacturer's suggested protocol.

Immunomagnetic Separation (IMS) E. coli E

Escherichia coli were isolated from aqueous suspensions by affinitycapture using the immunomagnetic beads. Suspensions of bacteria wereprepared in 1.5 mL microcentrifuge tubes (Brinkmann Instruments, Inc.,Westbury, Ny) by combining 100 μL of broth media with 900 μL ofphosphate buffer saline (PBS, 0.01 M Na2 HPO₄, 0.15M NaCl titrated to pH7.35 with HCl). Cell concentrations were determined using aPetroff-Hauser counting chamber (Hauser Scientific, Horsham, Pa.).

The immunomagnetic separation (IMS) procedure developed in thisinvestigation involved the following steps: In the first step, a 30 μLaliquot of the immunomagnetic beads were added to the bacterial samplesolution and incubated for 20 minutes at room temperature withcontinuous shaking. The second step involved concentrating the beads tothe side of the sample tube using a magnetic particle concentrator(Dynal, Lake Success, N.Y.) and removing the supernatant using a 1 mLpipette. In the third step, the magnet was removed and the beads werere-suspended in 1 mL of fresh PBS with vigorous shaking for 20 sec towash away any nonspecifically adhering components. The bead suspensionwas then transferred to a new tube and steps 2 and 3 repeated one moretime. In the fourth and final step, the beads were isolated with themagnet followed by decanting the buffer wash to waste and re-suspendingthe beads in 500 μL of deionized water. Subsequently, the bead-E. colicomplexes were admixed with a low titer (below the detection limit ofthe mass spectrometer) of the MS2 bacteriophage and incubated at roomtemperature with gentle shaking for 40 minutes. An aliquot of thesuspension was then removed and analyzed for the MS2 capsid proteinusing a sandwich sample preparation with a ferulic acid matrix (12.5 mgof ferulic acid in 1 mL of 17% formic acid: 33% acetonitrile: 50%deionized H₂O).

MALDI-TOF MS

All mass spectra were generated on a Voyager-DE STR+(AB AppliedBiosystems, Framingham, Mass.) MALDI-TOF mass spectrometer, operating inthe positive linear mode. The following parameters were used:accelerating voltage 25 kV, grid voltage 92% of accelerating voltage,extraction delay time of 350 nsec, and low mass ion gate set to 4 kDa.The laser intensity (N₂, 337 nm) was set just above the ion generationthreshold and pulsed every 300 ns. Mass spectra were acquired from eachsample by accumulating 100 laser shots from five different sample spots(final spectrum=average of 5×100 laser shots).

It should be appreciated that to assure that the detection of abiomarker for the bacteriophage indicates that the target bacterium ispresent in the liquid solution, a concentration of “parent”bacteriophage is applied to the liquid solution that is below thedetection limit for the bacteriophage or biomarker for the bacteriophagefor whatever analysis technique is employed. This assures that if thebacteriophage or the biomarker for the bacteriophage is detected by theanalysis technique, the detectable concentration of the bacteriophage orbiomarker is attributable to progeny bacteriophage, i.e., bacteriophageresulting from the replication of the bacteriophage by the targetbacterium present in the liquid solution. In certain situations, the useof such a concentration of “parent” bacteriophage has a multiplicity ofinfection (“MOI”) (i.e., the ratio of the number of parent bacteriophageto the number of target bacterium) that is too low to produce asufficient concentration of bacteriophages or biomarkers for thebacteriophage for detection.

To overcome the drawbacks associated with a low MOI, a sufficiently highconcentration of “parent” bacteriophage is added to the liquid solution.In this case, the concentration of the “parent” bacteriophage added tothe solution may exceed the detection limit of whatever analysistechnique is employed to detect the bacteriophage or biomarker of thebacteriophage. Consequently, analysis of a liquid solution treated inthis manner could detect the “parent” bacteriophages that were added tothe solution, rather than the progeny bacteriophage resulting fromreplication by the target bacteria.

Consequently, another embodiment of the process applies a concentrationof “parent” bacteriophage to the solution that is capable of beingdistinguished from any progeny bacteriophage. In one embodiment, theparent bacteriophage (i.e., the bacteriophage initially applied to thesolution) are “tagged” so that whatever analysis technique is employedis inherently capable of distinguishing the parent bacteriophage orparent bacteriophage biomarkers from the progeny bacteriophage orbiomarkers for the progency bacteriophage. For example, if a massspectral analysis technique is employed, the parent bacteriophage are“tagged” with a substance that alters or shifts the mass spectrum of theparent bacteriophage relative to the progeny bacteriophage, which willnot inherit the “tag” from the parent bacteriophage. For example, abiotinylated bacteriophage is employed as a parent bacteriophage and hasa different mass spectrum than the progeny bacteriophage produced by thebiotinylated bacteriophage infecting target bacterium present in thesolution. Other “tags” can be employed for other types of analyticaltechniques.

In another embodiment, the parent bacteriophage possesses acharacteristic that allows the parent bacteriophage to be separated fromany of the progeny bacteriophage in the liquid solution prior toanalysis, thereby assuring that most, if not all of the bacteriophagepresent in the liquid solution after separation are progenybacteriophage resulting from the replication of the parent bacteriophageby target bacteria present in the liquid solution. In one embodiment,biotinylated bacteriophage are initially applied to the liquid solution.Biotinylated bacteriophage are highly attracted to strepavidin.Consequently, to separate the biotinylated bacteriophage from the liquidsolution a strepavidin probe is utilized. In one embodiment, thebiotinylated bacteriophage are attached to a strepavidin coated probeand the probe is placed in the liquid solution. In this case, separationof the biotinylated bacteriophage from the liquid solution after theincubation period is accomplished by removing the probe from the liquidsolution. In another embodiment, strepavidin-coated magnetic beads areapplied to the liquid solution. The biotinylated bacteriophages areattached to the strepavidin-coated magnetic beads prior to theapplication of the beads to the solution. Alternatively, the beads areused to pick up biotinylated bacteriophages that were previously addedto the solution and then separated from the liquid solution using amagnet. In yet another, embodiment a strepavidin coated probe (e.g., aslide) is applied to the liquid solution after the incubation period.The biotinylated bacteriophages adhere to the probe and then the probeis separated from the liquid solution. Regardless of the manner in whichthe biotinylated bacteriophages are separated from the liquid mixture,at least a portion of the solution is then subjected to analysis todetermine if the bacteriophage or a biomarker for the bacteriophage ispresent, which indirectly indicates that the target bacteria was presentin the solution.

1. A method for determining if a target bacterium is present in a liquidsuspension comprising: providing a liquid suspension that might containa target bacterium; commingling a first quantity of a bacteriophage andsaid liquid suspension to provide said bacteriophage with an opportunityto infect at least some of any target bacterium that are present in saidliquid suspension and multiply the number of bacteriophage in saidliquid suspension, wherein said first quantity of said bacteriophage isbelow the detection limit of a detection technology; and analyzing,following said step of commingling, at least a portion of said liquidsuspension using said detection technology to determine if a biomarkerfor said bacteriophage is present that indirectly indicates that saidtarget bacterium was present in said liquid suspension.
 2. A method, asclaimed in claim 1, wherein: said step of analyzing comprises performinga mass spectrum analysis.
 3. A method, as claimed in claim 1, wherein:said step of analyzing comprises performing a MALDI analysis.
 4. Amethod, as claimed in claim 1, wherein: said step of analyzing comprisesperforming a MALDI-TOF analysis.
 5. A method, as claimed in claim 1,wherein: said step of analyzing comprises performing an electro-sprayionization mass spectrometry analysis.
 6. A method, as claimed in claim1, wherein: said step of analyzing comprises performing an ion mobilityspectrometry analysis.
 7. A method, as claimed in claim 1, wherein: saidstep of analyzing comprises performing an optical spectroscopy analysis.8. A method, as claimed in claim 1, wherein: said step of analyzingcomprises performing an immuno analysis.
 9. A method, as claimed inclaim 1, wherein: said step of analyzing comprises performing achromatographic analysis.
 10. A method, as claimed in claim 1, wherein:said step of analyzing comprises performing an aptamer analysis.
 11. Amethod, as claimed in claim 1, further comprising: determining whethersaid liquid suspension is likely to contain a biological element with abiomarker that could create a false positive for a biomarker for saidbacteriophage.
 12. A method, as claimed in claim 11, wherein: said stepof determining comprises assuming that a biological element is presentin said liquid suspension that has a biomarker that could create a falsepositive for a biomarker for said bacteriophage.
 13. A method, asclaimed in claim 11, wherein: said step of determining comprisesperforming an assay to determine if a biomarker is present that couldcreate a false positive for a biomarker for said bacteriophage.
 14. Amethod, as claimed in claim 11, further comprising: after adetermination has been made that said biological element is likely to bepresent, separating said target bacterium from said liquid suspension.15. A method, as claimed in claim 14, wherein: said step of separatingcomprises using immuno-magnet beads coated with antibodies for saidtarget bacterium.
 16. A method, as claimed in claim 14, furthercomprising: after a determination has been made, said step of separatingis performed prior to said step of commingling.
 17. A method, as claimedin claim 14, wherein: after a determination has been made that saidbiological element is likely to be present and after said step ofcommingling, separating said bacteriophage from said liquid suspension.18. A method, for determining if a target bacterium is present in aliquid suspension comprising: providing a liquid suspension that mightcontain a target bacterium; commingling a first quantity of abiotinylated bacteriophage and said liquid suspension to provide saidbiotinylated bacteriophage with an opportunity to infect at least someof any target bacterium that are present in said liquid suspension andproduce progeny bacteriophage in said liquid suspension; isolating,using a strepavidin coated surface, a substantial portion of saidbiotinylated bacteriophage from at least some of any of said progenybacteriophage produced during said step of commingling and therebyproduce an isolated liquid suspension that is substantially free of saidbiotinylated bacteriophage; and analyzing, following said step ofisolating, at least a portion of said isolated liquid suspension todetermine if a biomarker for said progeny bacteriophage is present thatindirectly indicates that said target bacterium was present in saidliquid suspension; said step of isolating comprises bringing saidstrepavidin coated surface and said liquid suspension into contact withone another to capture biotinylated bacteriophage in said liquidsuspension.
 19. A method, as claimed in claim 18, wherein: said step ofisolating comprises separating said strepavidin coated surface withattached biotinylated bacteriophage and a sample of said isolated liquidsuspension.
 20. A method, as claimed in claim 19, wherein: said step ofassaying comprises performing a mass spectrum analysis.